Medical Microbiology I - Lecture1

MEDICAL MICROBIOLOGY I

Lecture 1Lecture 1

Laboratory Safety and Bacterial

Growth

Laboratory Safety

Identify and evaluate hazards

Type of hazard:

1. Biohazards (infectious agents or items contaminated with them)contaminated with them)

2. Irritants (cause reversible inflammation)

3. Corrosive chemicals

4. Sensitizers (cause allergic reactions)

5. Carcinogens (induce tumours)

Laboratory Safety Guidelines

Wash hands with disinfectant soap when you arrive at the lab and again before you leave.

No eating, drinking, chewing gum, or smoking in the lab.

Do not put anything in your mouth such as pencils, Do not put anything in your mouth such as pencils, pens, labels, or fingers.

Avoid loose fitting items of clothing.

Application of cosmetics other than hand lotion likewise has no place within laboratory setting.

Keep your workspace free of all unnecessary materials.


Disinfect work areas before and after use with 70% ethanol or other disinfectant.

Label everything clearly.

Replace caps on reagents, solution bottles, and Replace caps on reagents, solution bottles, and bacterial cultures.

Do not open Petri dishes in the lab unless absolutely necessary.

Inoculating loops and needles should be flame sterilised in a Bunsen burner before you lay them down.


Turn off Bunsen burners when not in use.

Solutions must never be pipetted by mouth.

Treat all microorganisms as potential

pathogens. Use appropriate care and do not pathogens. Use appropriate care and do not

take cultures out of the laboratory.

Do not pour anything down the sink.

If hazardous powders have been handled, wash

around your nose and mouth so that adherent

particles are not ingested or inhaled.

Labeling

The solutions created in laboratory must be fully labeled.

Minimum information: Chemical name and, if a mixture, names of all Chemical name and, if a mixture, names of all

ingredients

Manufacturers name and address if purchased commercially, or person making the reagent

Date of purchase or made

Expiration date, if known

Hazard warnings and safety precautions

Personal Protective Equipment

Clothing suitable for laboratory work should be considered before protective equipment.

1. Secure, close-toed footwear

2. Lab coat 2. Lab coat

prevent biohazardous materials from reaching skin, and more importantly, any cuts, dermatitis, etc., that may be present

also protect street clothing from needing decontamination, as well as preventing contamination of laboratory cultures from the normal flora present on the skin


1. Aprons - disposable; heavy rubber aprons may be

needed when handling concentrated acids

2. Gowns - prevent biohazardous materials from

reaching skin and prevent contamination of

laboratory cultures

3. Goggles or safety glasses to protect the eyes by

preventing splashes into the eyes, nose and mouth,

and onto the skin

4. Full-face shields should be worn to protect the eyes,

nose and mouth, and the facial skin

Personal Protective Equipment5. Gloves

prevent exposure to microorganisms

glove material is rarely completely

impermeable; it delays penetration of harmful

material for a time sufficient to provide material for a time sufficient to provide

adequate protection

latex gloves are suitable

only for protection from

biohazards


For the best protection, the cuffs of the gloves should overlap the lower sleeves of the laboratory coat

Exam gloves must not be reused. They are designed for disposal after one use or if exposed to a chemical (they offer limited chemical protection).


6. Surgical masks-basic protection

7. Respirators

e.g. HEPA (high efficiency particulate air) mask-

better protection better protection

prevent the inhalation of aerosolized

microorganisms (inhalation exposure) when safety

equipment designed to contain infectious aerosols,

such as a biosafety cabinet, is not available

Specimen Handling

There are 3 potential routes of exposure:

1. Inhalation of aerosols

2. Contact with skin

3. Contact with mucous membranes (eyes, nose and 3. Contact with mucous membranes (eyes, nose and mouth)

Fresh specimens of human origin MUST always be considered potentially INFECTIOUS.

Fixed specimens have much-reduced risk because almost all infectious agents are deactivated by histological fixation.

Control of Hazardous Material

Proper control of hazardous material involves:

1. Collection

2. Transportation

3. Disposal / Decontamination 3. Disposal / Decontamination

Collection

Biohazardous material

Place discarded material in biohazard waste

container (lined with yellow biohazard bag).

Place used syringe / needle / glass slide / lancet Place used syringe / needle / glass slide / lancet

/capillary tube / broken glass fragments into

biohazard sharp container.

Discarded used microbiology plates must be

disposed in a biohazard container filled with

sodium hypochlorite.

Transportation

Laboratory personnel are responsible for the packaging of biohazardous / pathological waste.

If waste packaged in biohazard bags, have to be sealed / tiedsealed / tied

If waste are in container (biohazard / sharp), have to be closed and secured shut

All biohazard bags and container have to be labeled with the generators (lab) name, date and time.

They are also responsible for transporting the waste to the designated collection points.

Disposal / Decontamination

Depending on the material, there are several

means by which items can be treated.

The most common methods of treatment and

disposal are: disposal are:

Disinfection using chemicals

Sterilization using steam (autoclave)

Incineration (burning at high temperature)

Disinfection

Categorised as:

1. High level

2. Intermediate level

3. Low level

Effectiveness is influenced by

the nature of the item to be disinfected

number and resilience of the contaminating organism

amount of organic material present

type and concentration of disinfectant

duration and temperature of exposure

Disinfection

1. High-level disinfectants

e.g. moist heat, and use of liquids such as glutaraldelyde, hydrogen peroxide, peracetic acid, chlorine dioxide, and other chlorine compounds

Involve invasive procedures (e.g. certain types of Involve invasive procedures (e.g. certain types of endoscopes, surgical instruments with plastic or other components that cannot be autoclaved)

Cleaning the surface to remove organic material

Cleaning surface that are exposed to resilient organisms and bacterial spores

Disinfection

2. Intermediate-level disinfectants

e.g. alcohols, iodophor compounds, phenolic

compounds

Semi-critical instruments and devices such as flexible Semi-critical instruments and devices such as flexible

fiber-optic endoscopes, laryngoscopes, vaginal

specula, and anesthesia breathing circuits.

Clean surface or instruments in which contamination

with bacterial spores and other highly resilient

organisms is unlikely.

Disinfection

3. Low-level disinfection

e.g. quaternary ammonium compound

Used to treat non-critical instruments and devices

such as blood pressure cuffs, electrocardiogram such as blood pressure cuffs, electrocardiogram

electrodes, and stethoscopes

Although these items come into contact with

patients, they do not penetrate through mucosal

surfaces or into sterile tissues.

Disinfection

The level of disinfectants used for environmental surfaces is determined by the relative risk these surface pose as reservoir for pathogenic organisms.

High-level disinfectant should be used to clean High-level disinfectant should be used to clean the surface of instruments contaminated with blood

Not to clean surfaces that are dirty such as floors, sinks, and countertops.

Exception to this rule is if a particular surface has been implicated in nosocomial infection.

Disinfection

Chemical disinfection can also be achieved

using gas.

The most common example is the use of

ethylene oxide. ethylene oxide.

Gas disinfection is advantageous when the

sample is such that scrubbing of inner surfaces

cannot be done, such as in tubing.

Sterilization

Steam under pressure in an autoclave is a very effective form of sterilization.

High temperature (121 - 135C) for 15 min causes denaturation of microbial proteins.

Rapid rate but is influenced by: Rapid rate but is influenced by:

Temperature and duration of autoclaving

Size of the autoclave

Flow rate of the steam

Density and size of the load

Placement of load in the chamber

Incineration

Practiced routinely in microbiology laboratory to sterilise the inoculating loops.

Exposing the inoculating loop to a gas flame will burn up and vaporise any living microbes will burn up and vaporise any living microbes that are on the loop, ensuring that infectious organisms are not transferred.

Incineration is carried out in specially designed furnaces that achieve high temperatures and are constructed to be airtight.

Incineration

Proper incineration should:

occur very quickly

should not leave any residual material.

be smoke-free, otherwise microbes that are still be smoke-free, otherwise microbes that are still

living could be wafted away in the rising smoke

and hot air to cause infection elsewhere.

not too much sample as can result in an

incomplete burn.

Bacterial Growth

Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission.

Providing no mutational event occurs the Providing no mutational event occurs the resulting daughter cells are genetically identical to the original cell.

If the number surviving exceeds unity on average, the bacterial population undergoes exponential growth.

Bacterial Growth

1. Lag phase When microorganism are introduced into fresh culture

medium, usually no immediate increase in cell number.

Cells synthesising new components

The cells may be old, depleted of ATP, essential The cells may be old, depleted of ATP, essential cofactors, and ribosomes - all must be synthesised before growth can begin

New enzymes would be needed to use different nutrients.

Cells might be injured and need time to recover.

Inoculation of culture into a chemically different medium also results in a longer lag phase.

Bacterial Growth

Exponential or log phase

Microorganisms are growing and dividing at their

maximal rate and are influenced by:

Genetic potential

Nature of the medium Nature of the medium

Conditions under which they are growing

Rate of growth is consistent, so there is balanced

growth.

The population is most uniform in their chemical and

physiological properties.

Usually used in biochemical and physiological studies.

Bacterial Growth

Stationary phase In a closed system, eventually population growth

ceases and the growth curve becomes horizontal.

Attained by bacteria at a population level of around Attained by bacteria at a population level of around

10 cells / mL.

Final population size depends on:

nutrient availability

oxygen (other gases) availability

type of microorganism being cultured

Bacterial Growth

Stationary phase

Total number of viable microorganisms remain

constant due to:

balance between cell division and cell death, or balance between cell division and cell death, or

the population may simply cease to divide but remain

metabolically active.

Limiting factors

Nutrient

Oxygen / Other gases

Bacterial Growth

Senescence and Death

Assumed that detrimental environmental changes like

nutrient deprivation and the buildup of toxic wastes

caused irreparable harm resulting in loss of viability.

Even when bacterial cells were transferred to fresh

medium, no cellular growth was observed.

Loss of viability was often not accompanied by a loss

in total cell number, it was assumed that cell died but

did not lyse.

Viable but non-culturable (VBNC)

Programmed cell death or apoptosis

Growth of Microorganism

Several factors influence the growth of

microorganism:

1. Nutrition

2. Oxygen2. Oxygen

3. pH

4. Temperature

5. Moisture

Nutrition

Enters the cell after passing across the cells membrane.

Used by the cell for building material, cellular synthesis or for obtaining energy.

Nutritional requirements of bacteria varies among species.among species.

Some microorganism can obtain all their nutritional requirements from inorganic matter while others need many complex organic compounds.

Requirements: carbohydrates (sugar, starches and cellulose), a source of nitrogen, vitamins, water, and a source of energy

In addition to a proper physical

environment, microorganisms also

depend on an available source of depend on an available source of

chemical nutrients and are usually

grouped according to their energy and

carbon sources

Energy Source

1. Phototrophs

Use radiant energy (light) as their primary energy

source.

2. Chemotrophs

Use the oxidation and reduction of chemical

compounds as their primary energy source.

Carbon Source

Carbon is the structural backbone of the organic compounds that make up a living cell.

Based on their carbon sources, bacteria can be classified as:

1. Autotrophs

Require only carbon dioxide as a carbon source. An autotroph can synthesise organic molecules from inorganic nutrients.

2. Heterotrophs

Require organic forms of carbon. A heterotroph cannot synthesise organic molecules from inorganic nutrients.

Nutritional Pattern

Combining their nutritional patterns, all

organisms in nature can be placed into one of

four groups:

1. Photoautotrophs

2. Photoheterotrophs

3. Chemoautotrophs

4. Chemoheterotrophs

Photoautotroph

Photoautotrophs use light as an energy source and carbon dioxide as their main carbon source.

They include photosynthetic bacteria (green They include photosynthetic bacteria (green sulfur bacteria, purple sulfur bacteria and cyanobacteria), algae, and green plants.

Photoautotrophs transform carbon dioxide and water into carbohydrates and oxygen through photosynthesis.

Photoheterotroph

Photoheterotroph use light as an energy

source but cannot convert carbon dioxide into

energy.

Instead they use organic compounds as a Instead they use organic compounds as a

carbon source. They include the green non-

sulfur bacteria and the purple non-sulfur

bacteria.

Chemolithoautotroph

Chemolithoautotrophs use carbon dioxide as their main carbon source and inorganic compounds as an energy source.

Among the inorganic compounds are: Among the inorganic compounds are:

hydrogen sulfide

Sulfur

Ammonia

Nitrites

hydrogen gas

iron

Chemoorganoheterotroph

Chemoorganoheterotrophs use organic

compounds as both an energy source and a

carbon source.

Saprophytes live on dead organic matter while Saprophytes live on dead organic matter while

parasites get their nutrients from a living host.

Most bacteria, and all protozoas, fungi, and

animals are chemoorganoheterotrophs.

Nutrition

Minerals

1. Sulfur

Needed to synthesise sulfur-containing amino acids and certain vitamins.

Depending on the organism, sulfates, hydrogen Depending on the organism, sulfates, hydrogen sulfide, or sulfur-containing amino acids may be used as a sulfur source.

2. Phosphorus

Needed to synthesise phospholipids, DNA, RNA, and ATP.

Phosphate ions are the primary source of phosphorus.

Nutrition

3. Potassium, magnesium, calcium

Required for certain enzymes to function as well as additional functions.

4. Iron

Part of certain enzymes

5. Trace elements

Required in very minute amounts

Usually function as cofactors (electron donors or electron acceptors) in enzyme reactions

e.g. sodium, zinc, copper, molybdenum, manganese, and cobalt ions.

Oxygen

Microorganisms show a great variation in their requirements for gaseous oxygen.

Most can be placed in one of these groups:

1. Obligate aerobes

Organisms that only grow in the presence of oxygen. Organisms that only grow in the presence of oxygen.

Obtain their energy through aerobic respiration

2. Microaerophiles

Organisms that require a low concentration of oxygen (2-10%) for growth, but higher concentration are inhibitory.

Obtain their energy through aerobic respiration.

Oxygen

3. Obligate anaerobes

Organisms that grow only in the absence of oxygen

and are often inhibited or killed by its presence.

Obtain their energy through anaerobic respiration Obtain their energy through anaerobic respiration

or fermentation

4. Aerotolerant anaerobes

Like obligate anaerobes, they cannot use oxygen to

transform energy but can grow in its presence.

Obtain energy only by fermentation and are

known as obligate fermenters

Oxygen

5. Facultative anaerobes

Organisms that grow with or without oxygen, but

generally better with oxygen.

Obtain their energy through aerobic respiration if Obtain their energy through aerobic respiration if

oxygen is present, but use fermentation or

anaerobic respiration if it is absent.

Most bacteria are facultative anaerobes.

pH

Microorganisms can be placed in one of the

following groups based on their optimum pH

requirements:

1. Neutrophiles 1. Neutrophiles

Grow best at a pH range of 5 to 8

2. Acidophiles

Grow best at a pH below 5.5

3. Allaliphiles

Grow best at a pH above 8.5

Temperature

Bacteria have a minimum, optimum, and

maximum temperature for growth and can be

divided into 3 groups based on their optimum

growth temperature.growth temperature.

1. Psychrophiles

Cold-loving bacteria.

Their optimum growth temperature is between -

5 and 15C.

They are usually found in the Artic and Antarctic

regions and in streams fed by glaciers.

Temperature

2. Mesophiles

Bacteria that grow best at moderate temperatures.

Their optimum growth temperature is between 25 -

45C.

Most bacteria are mesophilic and include common soil

bacteria and bacteria that live in and on the body.

3. Thermophiles

Heat-loving bacteria.

Their optimum growth temperature is between 45 -

70C and are commonly found in hot springs and in

compost heaps.

Temperature

5. Hyperthermophiles

Bacteria that grow at very high temperatures.

Their optimum growth temperature is

between 70 - 110C. between 70 - 110C.

They are usually members of the Archae and

are found growing near hydrothermal vents at

great depths in the ocean.

Moisture

Moisture is required to:

carry food in solution into the cell,

carry waste in solution away from the cell, and

maintain the moisture content of the maintain the moisture content of the

cytoplasm.

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Medical Microbiology I - Lecture1